P65

EJC SUPPLEMENTS 13 (2015) 1– 75

42

N. Palkina*, T. Ruksha. Krasnoyarsk State Medical University named

A. Petukhov*, A. Daks, O. Shuvalov, N. Barlev. Institute of Cytology,

after Prof. V.F. Voino-Yasenetsky, Ministry of Public Health, Krasnoyarsk,

Russian Academy of Sciences, St. Petersburg, Russian Federation

Russian Federation

⇑

Corresponding author.

⇑

Corresponding author.

Matrix metalloproteinases (MMPs) – zinc containing endopep-

In 2004, COP1 was characterized as p53 E3-specific human

tidases which cleave the different components of the extracellu-

ubiquitin ligase according to in vitro studies. Later, oncogenic

lar matrix. However, not all biological mechanisms and the

activity of COP1 and correlation of its overexpression with level

effects of these endopeptidases are well-established. Previously

of p53 protein in clinical tumor samples and cell lines were

it was believed that MMP functions only as proteolytic enzymes,

shown. High expression level of COP1 was described in 25 of 32

but it was found later that MMPs could act as regulators of protein

cases of breast carcinomas and in 76 out of 171 cases of ovarian

kinase signal transduction pathways by modifying receptors and

carcinomas. High expression level of COP1 was described in hep-

signaling molecules.

atocellular carcinoma cell lines (PLC, Hep3B and HepG2, Huh7). In

One of the most important substrates for MMP is transforming

40 of 55 cases of pancreatic cancer, overexpression of COP1 was

growth factor-b (TGF-bÞ, which is a receptor for the ligand TGF-b-

also noted. Some types of leukemia, melanoma, breast, lung

signal transduction pathway comprises a network of protein

and prostate cancer contain focal deletions of COP1. COP1 overex-

kinase pathways responsible for many of the processes of tumor

pression was also found in non- oncological diseases like

progression, including epithelial mesenchymal transformation

Duchenne muscular dystrophy, ischemic cardiomyopathy and

(EMT). Despite the fact that melanoma cells are not of epithelial

juvenile dermatomyositis.

origin, they express the epithelial E-cadherin that is necessary

Similar to other p53-related E3 -ubiquitin ligases COP1 has dif-

for their interaction with the basal layers of the epidermis. The

ferent substrates. The selection of target for ubiquitination

degradation of the extracellular matrix and loss of expression of

depends on cell type or differentiation stage. Among the sub-

E-cadherin are major changes needed to start the processes of

strates of COP1, besides p53 are: c-Jun, ETV1, ACC, TORC2, FOXO1

melanoma invasion. One of the markers of EMT is a transcription

and C/EBPa, FIP200.

factor twist1. Twist1 activates the processes of epithelial-

To identify novel proteins that interact with COP1 we applied

mesenchymal transformation and increases the invasion of mel-

proteomics. Proteins co-immunoprecipitated with COP1 were

anoma cells by increasing transcription of MMP. The aim of this

analyzed by MALDI -TOF mass spectrometry. To achieve this,

study was to evaluate expression levels TGF-b, twist1 under selec-

COP1–3xFlag expression vector was generated and transfected

tive inhibition of MMP-9 and combined inhibition of MMP-9 and-

into HEK 293T cells by calcium phosphate transfection method.

13 in melanoma model in vivo; to determine the role of MMPs as

Protein complexes were immunoprecipitated with anti-FLAG

possible regulators of TGF-b-way signal transduction, as well as

beads. Bound proteins were then separated by 1D SDS–PAGE gel

associated with TGF-b EMT process.

electrophoresis and analyzed by means of ESI-LC/MS/MS mass

The study was approved by the local Ethic Committee (proto-

spectrometry. Subsequent bioinformatics analysis of the interact-

col No. 144/ 2012 of 15.11.2012). Melanoma B16-bearing mice (con-

ing proteins was employed. About 25% of identified proteins were

trol group consisted of 7 animals, MMP-9 inhibitor treatment

attributed to the cytoskeleton proteins, almost 20% of proteins

group – 7, MMP-9/13 inhibitor treatment group – 7 animals) under-

had known role in metabolism. According to the literature data,

went experimental treatment by MMP inhibitor (Calbiochem,

it was not surprising to find several proteins involved in the lipid

USA) by daily application within 7 days. Control group consisted

metabolism. Finally, a significant groups of interactants play role

of animals without treatment. Efficacy of MMP-9 inhibition was

in transcription, cell adhesion, apoptosis, protein modification

evaluated by gelatinize zymography. TGF-b1 and twist 1 tran-

and transport. It is important to note that several COP1-bound

scription factor expression was determined by PCR real-time

proteins can be strong regulators of cell cycle in G2/M transition.

using StepOne System (Applied Biosystems, USA). Statistical

Our data can be used for the subsequent studies of previously

analysis was done by Kruskal–Wallis test and Multiple Compar-

unknown roles of COP1 in different diseases.

isons. The P values lower 0.05 were considered as significant. It was found that the selective inhibition of MMP-9 did not induce changes in expression levels of TGF-b and twist1, however,

This work was funded by Grants from Russian Scientific Foundation – Russia (No. 114-15-00816) and Grant of Molecular and Cellular Biology of the Presidium of the Russian Academy of Sciences.

the combined inhibition of MMP-9 and MMP-13 significantly reduced the expression levels of the transcription factor twist1 and TGF-b. The results show that MMP-9 and MMP-13 act as acti-

http://dx.doi.org/10.1016/j.ejcsup.2015.08.075

vators of TGF-b-signal transduction pathway and could be considered as potential molecular targets for experimental therapy for cancer. http://dx.doi.org/10.1016/j.ejcsup.2015.08.074

P65

Development of allele-specific PCR assays for detection of mutations in KRAS gene in colorectal cancer in Russian patients

P50

E. Pisarevaa,b,*, N. Gutkinaa, S. Kovalenkoa,b, V. Shamanina,b. a

Detection of COP1-interacting proteins using co-immunoprecipitation followed MALDI-TOF mass spectrometry

Institute of molecular biology and biophysics, Novosibirsk, Russian

Federation, ⇑

b

Biolink

Ltd.,

Corresponding author.

Novosibirsk,

Russian

Federation

43

EJC SUPPLEMENTS 13 (2015) 1– 75

KRAS is component of Ras/MAPK signaling cascade that regu-

New assays have high sensitivity and specificity and are suit-

lates cell proliferation and cell survival. Somatic mutations in

able for detection of KRAS mutations in clinical FFPE tumor

KRAS gene are often found in tumors and affect the sensitivity

samples.

of tumors to target therapy. Mutations in codons 12 or 13 of KRAS gene in colorectal cancer (CRC) are associated with resistance to anti-EGFR

antibodies

Cetuximab

and

Panitumumab.

http://dx.doi.org/10.1016/j.ejcsup.2015.08.076

The

objectives of this work were to develop PCR tests for detection mutations in KRAS gene and analyze the frequency of mutations

P90

in KRAS gene in CRC in Russia. DNA sequencing by Sanger is the most common method for mutation analysis. However, the method has a sensitivity of

Dynamic changes of circulating microRNA expression in response to

20% mutant allele, which is often not sufficient for the analysis

the lung cancer combined therapy

of somatic mutations in tumors. One of the most sensitive mutation analysis methods is allele-

A. Ponomaryovaa,c,*, E. Rykovab, N. Cherdyntsevaa,d, E. Morozkinb,

specific real-time PCR. This method allows to detect 1% of

I. Zaporozhchenkob, T. Skvortsovab, A. Dobrodeeva, A. Zav’yalova,

mutated DNA in the sample. This sensitivity is sufficient for anal-

S. Tuzikova, V. Vlassovb, P. Laktionovb.

ysis of mutations in tumor samples containing 2–5% or more of tumor cells in normal tissue. In this study we developed and compared 3 new KRAS assays

a

Tomsk Cancer Research

b

Institute, Tomsk, Russian Federation, Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, Russian Federation, c

National Research Tomsk Polytechnic University, Tomsk, Russian d

National Research Tomsk State University, Tomsk,

(1) real-time PCR with allele-specific primers; (2) real-time wild-

Federation,

type blocking PCR with LNA (locked nucleic acid) blocker; and

Russian Federation

⇑

Corresponding author.

(3) Sanger sequencing with LNA-blocker. First assay is a PCR test with seven reactions using allele-specific primers for detection

Background:

and genotyping 7 mutations in 12 and 13 codons of KRAS gene.

(DNAs and RNAs) in the blood seems to be a promising approach

The analysis of circulating tumor nucleic acids

Second assay is real-time PCR with only a single pair of primers

for the development of the low-invasive methods of tumor detec-

and LNA oligonucleotide blocker. LNA-blocker is an oligonu-

tion, valuable for clinical practice. Detection of oncogenic and

cleotide which has a wild-type sequence of codons 12 and 13

tumor suppressor miRNAs in the blood plasma/serum is evidence

of KRAS gene. LNA-blocker binds strongly to wild-type KRAS

of their participation in pathogenesis and suggest the possibility

DNA and suppresses its amplification, but does not block ampli-

of their use as tumor markers and targets for therapy. Thus, the

fication of mutant DNA. The real-time PCR with LNA -blocker

determination of expression level of tumor-associated miRNAs

can detect mutant DNA but does not genotype mutation. Such

in plasma can lead to significant progress in understanding the

assay can be used as a simple and sensitive screening test for

tumor development and treatment.

mutant KRAS cases if exact genotyping of mutation is not

Aim:

required. We also used LNA-blocker to increase sensitivity of

(miR-19b, miR-25, miR-125b, miR-126, miR-205) in blood plasma

Sanger sequencing. To evaluate sensitivity and specificity of

from lung cancer patients during combined therapy and to

new tests DNA standards were prepared with different ratios of

estimate their value as disease monitoring markers.

normal and mutant alleles using normal human DNA without

Materials and methods:

mutation and recombinant plasmids with mutations in KRAS

patients (n = 23) with non-small cell lung cancer treated at the

(G12C, G12S, G12R, G12V, G12D, G12A, G13D). After optimization

Tomsk Cancer Research Institute. These samples were stabilized

all three assays had sensitivity 5% of mutant alleles for the

and fractionated into plasma and blood cells. MicroRNA was iso-

detection of mutations in KRAS gene, using 2.5–40 ng of human

lated from blood plasma using single-phase phenol-free extrac-

DNA.

tion protocol and purified on silica-based spin columns

Estimation the changes in expression level of miRNAs

Blood samples were taken from

Performance of new assays for KRAS mutations was compared

(BioSilica Ltd, Novosibirsk, Russia). Concentration of five above

using 81 colorectal tumor samples. Before analysis relative con-

mentioned miRNAs was measured by quantitative RT-PCR and

tent of tumor cells in the samples was evaluated by pathologist.

normalized to miR-16 using dCt method.

If tumor content was less than 20% in the sample then regions

Results:

with a maximum number of tumor cells were manually

changes of circulating DNA in blood plasma from lung cancer

macrodissected before the DNA extraction. DNA was purified

patients during the combined therapy. Circulating miRNAs were

from formalin fixed paraffin embedded (FFPE) tissue using

isolated from plasma samples of non-small cell lung cancer

‘‘FFPE-DNA Kit” (Biolink). All three assays had high sensitivity

patients before treatment, within 30 days after completing

(95–100%) and specificity (100%) for detection of KRAS mutations

chemotherapy and 15 days after surgery, by using developed

in clinical tumor samples. Mutations of the KRAS gene were

methodological approach. In case of miR-19b and miR-125b anal-

found in 37 of 81 cases (46%) of CRC including 12 cases of muta-

ysis was found that the miRNA expression level correlates with

tion G13D, 11 cases of G12D, 5 cases of mutation G12V, 4 cases

clinical response to chemotherapy and surgery. Increasing level

G12C, 3 cases of mutation G12A, 2 cases G12S, 1 case G13R. A sin-

of miR-19b and decreasing level of miR-125b were associated with

gle case with mutation G13R was missed by allele-specific PCR but

therapeutic response. Using Repeated measures ANOVA analysis

was detected by real-time PCR with LNA-blocker and confirmed

we demonstrated that the miR-19b and miR-125b expression

by sequencing.

levels changes throughout three check-up points during the

In this study we analyzed the dynamic expression